The faithful transmission of genetic information from one cell generation to the next is a fundamental property of all living systems, and in humans, it represents a major barrier against disease. To avoid genome instability, cells have evolved numerous DNA repair pathways, and they strictly limit DNA replication to a single round per cell cycle. In the last two funding perios, we used Xenopus egg extracts to characterize a novel E3 ubiquitin ligase called CRL4Cdt2. CRL4Cdt2 substrates contain a PIP degron that mediates binding to the DNA polymerase processivity factor, PCNA, on DNA. Once this binary complex of PCNA and substrate has formed, CRL4Cdt2 is recruited to generate a ternary complex, and substrate ubiquitylation takes place on chromatin. The dependence of CRL4Cdt2 activity on DNA-bound PCNA (PCNADNA) insures that substrates are destroyed only in S phase and after DNA damage. In vertebrates, CRL4Cdt2 promotes the S phase destruction of at least three proteins (Cdt1, p21, and Set8) that control origin firing during the cell cycle. Given its central role as a custodian of the genoe, it will be crucial to determine the molecular mechanism of how CRL4Cdt2 recognizes its substrates, in particular how this recognition is coupled to PCNADNA. Indeed, CRL4Cdt2 is the only known ubiquitin ligase that recognizes substrates when they are displayed on another polypeptide. As such, studying its mechanism has the potential to establish new paradigms for regulated proteolysis. Because the identification of each new CRL4Cdt2 substrate has lent important insights into the proces of genome maintenance, identifying additional targets is also a top priority. In this proposal, we will: (1) characterize a new CRL4Cdt2 substrate involved in DNA repair and use proteomics to discover other CRL4Cdt2 targets. (2) Use a novel, extract-based single molecule approach to determine how ubiquitylated substrates dissociate from PCNA so that a new substrate may bind. (3) Address how many subunits of PCNA are required to support CRL4Cdt2 activity and DNA replication. (4) Elucidate how CRL4Cdt2 activity is coupled to DNA-bound PCNA. Together, the experiments will explore the biology and mechanism of a new S phase-specific proteolysis pathway that acts as an essential custodian of genome integrity. PUBLIC HEALTH RELEVANCE: Narrative To maintain the integrity of our genomes, it is crucial that cells make one precise copy of their DNA before each cell division (DNA replication). Previously, we discovered that this regulation involves the destruction of a key replication initiation factor called Cdt1. Destructionis triggered by a highly unusual ubiquitin ligase called CRL4Cdt2 that targets specific proteins for destruction. In this proposal, we expand our search for new targets of this ligase and we explore the molecular mechanism by which it recognizes its substrates.